Clathrin-coated vesicles (CCV) are intracellular devices for membrane traffic from the cell surface and the trans-Golgi network to endosomes. They are not only important universal vehicles used by all nucleated cells for intracellular membrane traffic, but also have tissue-specific functions, especially in neurons and in the immune system. CCV pathways are involved in metabolic diseases, neurological disorders and viral infections. A critical step in the CCV "life cycle" is clathrin-lattice uncoating, a process mediated by the chaperone Hsc70 and co-chaperone auxilin. I propose to determine the molecular mechanism of the uncoating process, through structural studies and biochemical experiments. I will obtain higher resolution cryoEM structures of clathrin coats of different sizes and designs, to understand the role of invariant interactions in stabilizing the clathrin lattice. I will then determine the positions of Hsc70 and auxilin in these lattices, to visualize how uncoating starts. I will test hypotheses through studies of assembly and uncoating of cages formed from recombinant clathrin bearing specific mutations. I will also attempt to obtain crystals of Hsc70 bound with the auxilin J-domain and of the auxilin PTEN-like domain, to understand the function of auxilin through its x-ray crystal structures. These studies will elucidate the molecular mechanism of Hsc70/auxilin-mediated CCV uncoating and greatly advance understanding of CCV pathways. My work will also provide a general framework for using modern X-ray crystallography and cryoEM techniques to acquire high-resolution information on intricate molecular machines. Clathrin-coated vesicles play essential roles in maintaining the health of neurons and immune system. Defects in CCV pathways correlate with a broad spectrum of human diseases, including familial hypercholesterolemia, leukemias, Alzheimer's disease, influenza and HIV. Understanding steps in CCV life cycle as proposed by this research will assist in the discovery of new therapeutic strategies for diseases involving defects in membrane traffic.